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¹ Central Research Laboratory (Nakahari Project)² Department of Internal Medicine (Division of Gastroenterology)³ Department of Microbiology⁴ Department of Physiology⁵ Department of Pathology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, Japan

	Abstract

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Prostaglandin E₂ (PGE₂), which is generated by two isoforms of cyclo-oxygenase (COX₁ and COX₂), is a key mediator in gastric mucosal defense. In the present study, antral mucosa of guinea-pigs was incubated with various agonists or antagonists in a medium, the PGE₂ concentration of which was measured using a PGE₂ EIA kit. Prostaglandin E₂ was released from the antral mucosa spontaneously (basal PGE₂ release) and acetylcholine (ACh, 10 µM) enhanced the PGE₂ release (ACh-stimulated PGE₂ release) was mediated via intracellular Ca²⁺ concentration ([Ca²⁺]_i). Arachidonic acid enhanced both forms of PGE₂ release, and a phospholipase A₂ inhibitor (amylcinnamoyl anthranilic acid) and COX inhibitors (acetylsalicylic acid and indomethacin) decreased them. 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol (SC560, 100 nM, a COX₁-selective inhibitor) inhibited ACh-stimulated PGE₂ release without any decrease in basal PGE₂ release. N-(2-Cyclohexyloxy-4-nitrophenyl) methanesulphonamide (NS398, 20 µM, a COX₂-selective inhibitor) decreased basal PGE₂ release without any reduction of ACh-stimulated PGE₂ release. However, ionomycin (a Ca²⁺ ionophore) increased PGE₂ release from antral mucosa in the presence of SC560 or NS398, suggesting that COX₁ and COX₂ are regulated by [Ca²⁺]_i. These findings indicate that COX₁-containing cells have ACh receptors but COX₂-containing cells do not. Moreover, in isolated antral epithelial cells, SC560 decreased basal and ACh-stimulated PGE₂ release, but NS398 did not. In conclusion, in antral mucosa, basal PGE₂ release is mainly maintained by COX₂ of non-epithelial cells, and ACh-stimulated PGE₂ release is maintained by COX₁ of epithelial cells.

(Received 5 May 2006; accepted after revision 29 August 2006; first published online 31 August 2006)
Corresponding author T. Nakahari: Department of Physiology, Osaka Medical College, 2–7 Daigaku-cho, Takatsuki 569-8686, Japan. Email: takan{at}art.osaka-med.ac.jp

	Introduction

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Prostaglandin E₂ (PGE₂) regulates various gastric cytoprotective actions, including acid secretion, mucin synthesis, mucin secretion, HCO₃^– secretion, blood flow and gastric motility (Wallace, 1992; Yoshimura et al. 1992; Peskar et al. 2001; Simmons et al. 2004; Takeeda et al. 2004). Of these actions, mucin synthesis and secretion are of particular importance in view of mucosal surface protection (Wallace, 1992; Yoshimura et al. 1992). Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit PGE₂ synthesis, can induce gastric lesions, and supplementation with PGE₂ prevented NSAIDs-induced lesions (Wallace, 1992; Hawkey, 2000; Peskar et al. 2001; Simmons et al. 2004). Thus, PGE₂ plays a key role in gastric mucosal defence.

Prostaglandin E₂ is synthesized in gastric mucosa (Yoshimura et al. 1992; Hawkey, 2000; Peskar et al. 2001; Simmons et al. 2004; Takeeda et al. 2004; Shimamoto et al. 2005). We have demonstrated that PGE₂ activates exocytosis in isolated antral mucous cells, mediated by cAMP accumulation via EP4 receptors (Ohnishi et al. 2001), and enhances Ca²⁺-regulated exocytosis in ACh-stimulated antral mucous cells (Shimamoto et al. 2005). However, no exocytotic events were observed in unstimulated antral mucous cells. This suggests that PGE₂ is unlikely to be released from unstimulated isolated antral mucous cells (Fujiwara et al. 1999, 2006; Ohnishi et al. 2001; Nakahari et al. 2002; Shimamoto et al. 2005).

Shimamoto et al. (2005) reported that PGE₂ was released from antral mucosa spontaneously, and the concentration of PGE₂ was estimated to be approximately 50–60 nM in antral mucosa. Prostaglandin E₂ (50–60 nM) was reported to stimulate cAMP-regulated exocytosis in antral mucous cells (Ohnishi et al. 2001). These findings appear to be inconsistent with those demonstrating that antral mucous cells do not release PGE₂ without ACh stimulation (Shimamoto et al. 2005). These differences between the reports may be result from differences in sample preparations; that is, antral mucosa and antral mucous cells. Non-epithelial cells may release PGE₂ in unstimulated antral mucosa.

Prostaglandins are synthesized by two isoformes of cyclo-oxygenase (COX): COX₁ and COX₂. Biochemical examinations demonstrated that COX₁ protein exists in intact gastric mucosa, but COX₂ protein does not (Gretzer et al. 1998, 2001; Tatsuguchi et al. 2000; Peskar et al. 2001). Immunohistochemical examinations also demonstrated that COX₁ is distributed in epithelial cells, but COX₂ is not detected in normal gastric mucosa (Iseki, 1995; Sawaoka et al. 1998; Sun et al. 2000; Takeeda et al. 2004; Tanigawa et al. 2004). However, COX₂ immunoreactivity was induced in interstitial cells, such as macrophages and fibroblasts, by the intragastric instillation of HCl, ulceration and inflammation (Iseki, 1995; Sawaoka et al. 1998; Sun et al. 2000; Tatsuguchi et al. 2000; Peskar et al. 2001; Miyake et al. 2002; Tanigawa et al. 2004). Moreover, COX₂ generates a small proportion of the gastric prostaglandins (Shigeta et al. 1998), and no gastric lesions are observed in COX₂-deficient mice (Peskar et al. 2001; Simmons et al. 2004). These findings appear to suggest that COX₁ synthesizes prostaglandins in the normal gastric mucosa and that COX₂ synthesizes them in the injured gastric mucosa, such as gastric ulcers and inflammatory lesions.

In contrast, recent reports demonstrated that COX₂-selective inhibitors delay the repair of gastric ulcers in experimental animals, and suppress gastric cell proliferation and angiogenesis (Wallace et al. 2000; Gretzer et al. 2001), and that COX₂ in the kidney and cardiovascular system maintains their functions (McAdam et al. 1999). These observations indicate that, in contrast to the initial concept, COX₂ plays an important role in maintaining gastric mucosal integrity.

Our previous studies demonstrated that the amounts of PGE₂ released can be measured in stripped antral mucosa (Shimamoto et al. 2005; Fujiwara et al. 2006). In the present study, we examine the effects of various COX inhibitors on PGE₂ release to clarify which COX subtypes, COX₁ or COX₂, synthesize PGE₂ in the intact antral mucosa.

	Methods

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Solutions and chemicals

Solution I contained (mM): NaCl, 121; KCl, 4.5; NaHCO₃, 25; MgCl₂, 1; CaCl₂, 1.5; Na-Hepes, 5; H-Hepes, 5; and glucose, 5. To prepare a Ca²⁺-free solution, CaCl₂ was excluded from solution I and EGTA (1 mM) was added. The pH values of the solutions were all adjusted to 7.4 by adding HCl (1 M). The solutions were aerated with a gas mixture (95% O₂ and 5% CO₂) at 37°C. Ionomycin, thapsigargin, indomethacin (IDM) and acetylsalicylic acid (ASA), were purchased from Sigma (St Louis, MO, USA). Acetylcholine chloride (ACh), collagenase and bovine serum albumin (BSA) were purchased from Wako (Osaka, Japan). N-(p-Amylcinnamoyl) anthranilic acid (ACA), 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol (SC560) and N-2-cyclohexyloxy-4-nitrophenyl methanesulphonamide (NS398) were purchased from Merck Biosciences (Germany). All the reagents were dissolved in dimethyl sulphoxide (DMSO) and were prepared to their final concentrations immediately before the experiments. The concentration of DMSO did not exceed 0.1%, and this concentration does not have any effect on cellular actions (Fujiwara et al. 1999, 2006; Ohnishi et al. 2001; Nakahari et al. 2002; Hayashi et al. 2005; Murao et al. 2005).

Cell preparation

Hartley strain male guinea-pigs weighing approximately 250 g were purchased from Shimizu (Kyoto, Japan), and were maintained on standard pelleted food and water. The guinea-pigs were anaesthetized by intraperitoneal injection of pentobarbitone sodium (60–70 mg kg^–1), after which they were killed by cervical dislocation. All experimental procedures were approved by the Animal Research Committee of Osaka Medical College, and the animals were cared for according to the guidelines of this committee. The procedures for the cell preparation have previously been described in detail (Fujiwara et al. 1999, 2006; Ohnishi et al. 2001; Nakahari et al. 2002; Shimamoto et al. 2005). Briefly, the gastric antrum was excised, and the mucosal layer was stripped from the muscle layer using glass slides. For the experiments on isolated antral epithelial cells, the stripped antral mucosa was digested with 0.1% collagenase for 10 min at 37°C in solution I containing 2% BSA. The digested mucosa was then filtered through a nylon mesh with a pore size of 150 µm squares and then washed with solution I containing 2% BSA three times, with centrifugation (approximately 20g for 1 min) between each wash. The cells were resuspended in solution I (4°C).

Measurement of PGE₂ concentration

The stripped antral mucosa was weighed and then stored in the solution I at 4°C before the start of experiments (30–60 min). After a 3 min warm-up period, the stripped antral mucosa was incubated in the control solution (10 ml) aerated with a gas mixture (95% O₂ and 5% CO₂) for 10 min at 37°C, prior to ACh stimulation, and then incubated for a further 15 min. In the vehicle control group, DMSO (10 µl) was added instead of ACh (10 µM). When inhibitors were used, the antral mucosa, after the 3 min warm-up period, was incubated with the inhibitors for 10 min prior to ACh stimulation. Five hundred microlitres of the incubation solution was transferred to a microtube immediately before and 2, 5, 10 and 15 min after ACh stimulation. The microtube containing the sample was immediately cooled on ice, and stored at –30°C until the PGE₂ concentration measurements. Prostaglanding E₂ concentations were measured using a PGE₂ enzymeimmunoassay (EIA) kit (no. 514010, Cayman, Ann Arbor, MI, USA), and PGE₂ contents were expressed as micrograms per gram wet weight of tissue (µg (g tissue)^–1; Welch et al. 2003; Shimamoto et al. 2005; Fujiwara et al. 2006). The amount of PGE₂ released was calculated from the difference between the values before and 2, 5, 10 and 15 min after ACh stimulation.

In experiments on isolated cells, strips of antral mucosa from two or three guinea-pigs were digested with 0.1% collagenase for cell isolation. The isolated cells were suspended in solution I (10 or 15 ml at 4°C). The cell suspension was divided into two or three test tubes (2 or 3 aliquots of 5 ml) and then stored at 4°C until the start of experiments. In the control experiments, two test tubes were used. After a 2 min warm-up period, the cell suspension was aerated with a gas mixture (95% O₂ and 5% CO₂) at 37°C for 10 min. Acetylcholine chloride (10 µM) or DMSO (10 µl) was added into each test tube, and then the cells were incubated for a further 15 min. In the experiments using inhibitors, three test tubes were used. After a 2 min warm-up period, two test tubes were incubated with the inhibitors for 10 min. Acetylcholine chloride (10 µM) was added into the one test tube, and DMSO was added into the other test tube. The third test tube was used as the vehicle control. After the 2 min warm-up period, the cell suspension was incubated at 37°C for 10 min, prior to DMSO addition, and then incubated for a further 15 min.

After brief centrifugation (20g, 30 s), 450 µl of the incubation solution was transferred to a microtube before and 15 min after ACh stimulation. The microtube containing the sample was immediately cooled on ice, and stored at –30°C until the assay for PGE₂ content. The amount of PGE₂ was calculated from the difference between the values before and 15 min after ACh stimulation. To compare the amount of PGE₂ released in each experiment, the increase in the amount of PGE₂ (%) was calculated by comparison with each vehicle control value.

The statistical significance of the difference between mean values was assessed using Student's paired or unpaired t test as appropriate. Differences were considered significant at P < 0.05.

	Results

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Figure 1 shows light micrographs of stripped mucosa and isolated epithelial cells, which were stained using Haematoxylin and Eosin (Fig. 1A and B) and Periodic acid–Shiff (PAS; Fig. 1C and D). The mucosal strips were used for the experiments on antral mucosa (Fig. 1A and C), and the collagenase-digested antral glandular columns were used for the experiments on isolated epithelial cells (Fig. 1B and D). Isolated glandular columns contain pyloric gland cells, some of which contain PAS-positve granules (Fig. 1D). As shown in Fig. 1B and D, non-epithelial cells, such as vascular cells, smooth muscle cells, blood cells and fibroblasts, were present only in numbers. The effects of non-epithelial cells appear to be negligibly small.

Effects of ACh

In unstimulated antral mucosa, PGE₂ was released spontaneously (Fig. 2A), and the amount of PGE₂ released during 15 min was 0.112 ± 0.020 µg (g tissue)^–1 (n = 4). Stimulation with ACh (10 µM) enhanced PGE₂ release from antral mucosa (Fig. 2A), and the amount of PGE₂ released over 15 min (ACh-stimulated PGE₂ release) was 0.289 ± 0.016 µg (g tissue)^–1 (n = 4). Addition of the vehicle control (DMSO) alone did not enhance PGE₂ release from antral mucosa, and the amount of PGE₂ released over 15 min (basal PGE₂ release) was 0.136 ± 0.006 µg (g tissue)^–1 (n = 5; Fig. 2A). To compare PGE₂ release among experiments, we used the amount of PGE₂ released during the 15 min from the start of the experiments. The value of basal PGE₂ release was taken to be the amount of PGE₂ released during 15 min with addition of the vehicle control (DMSO).

The effects of ACh dose on PGE₂ release were examined. Acetylcholine increased the amount of PGE₂ released in a dose-dependent manner (Fig. 2B). The amounts of PGE₂ released over 15 min during ACh stimulation were 0.153 ± 0.014 µg (g tissue)^–1 (n = 3) at 0.1 µM and 0.190 ± 0.008 µg (g tissue)^–1 (n = 5) at 1 µM, and reached a plateau level (approximately 0.3 µg (g tissue)^–1) at ACh concentrations higher than 10 µM. Based on these observations, the concentration of ACh used was 10 µM throughout the subsequent experiments. The amounts of PGE₂ released by stimulation with ACh concentrations higher than 1 µM were significantly increased compared with those in unstimulated mucosa (P < 0.05).

Effects of intracellular Ca²⁺ concentration ([Ca²⁺]_i)

The actions ACh are mediated by an increase in [Ca²⁺]_i in antral mucous cells (Fujiwara et al. 1999, 2006; Nakahari et al. 2002; Shimamoto et al. 2005). The effects of [Ca²⁺]_i on PGE₂ release were examined in antral mucosa (Fig. 3). Ionomycin (IM, 1 µM) and thapsigargin (TG, 2 µM), which increase [Ca²⁺]_i in antral mucous cells (Shimamoto et al. 2005), enhanced PGE₂ release. The amounts of PGE₂ released in IM-stimulated antral mucosa were greater than those in ACh-stimulated antral mucosa (P < 0.05).

The effects of Ca²⁺-free solutions on PGE₂ release were examined in unstimulated and ACh-stimulated antral mucosa. In control conditions, the amount of PGE₂ released in the Ca²⁺-free solution was 0.032 ± 0.006 µg (g tissue)^–1 (n = 3), which was approximately 25% of that in the Ca²⁺-containing solution (solution I), and ACh enhanced PGE₂ release slightly but significantly (0.100 ± 0.005 µg (g tissue)^–1, n = 3). However, the amount of PGE₂ released during ACh stimulation in the Ca²⁺-free solution was approximately 35% of that in solution I. In Ca²⁺-free solution, ACh has been shown to increase [Ca²⁺]_i by stimulating Ca²⁺ release from intracellular stores in antral mucous cells (Fujiwara et al. 1999; Nakahari et al. 2002; Shimamoto et al. 2005). To chelate intracellular Ca²⁺, the antral mucosa was incubated in the Ca²⁺-free solution containing BAPTA AM (25 µM; Ohnishi et al. 2001). In the presence of BAPTA AM, ACh did not enhance PGE₂ release (0.034 ± 0.007 µg (g tissue)^–1, n = 3). Thus, the maintenance of low [Ca²⁺]_i eliminated the enhancement of PGE₂ release in ACh-stimulated mucosa.

Effects of COX inhibitors

The effects of non-selective COX inhibitors, acetylsalicylic acid (ASA, 10 µM) and indomethacin (IDM, 10 µM), were examined (Fig. 4A). Acetylsalicylic acid (10 µM) added prior to the vehicle addition decreased the amount of PGE₂ released by 75% (n = 3) in unstimulated mucosa, and in the presence of 10 µM ASA the addition of ACh did not enhance PGE₂ release. Similar experiments were performed using IDM (10 µM). The addition of IDM (10 µM) decreased the amount of PGE₂ released in the presence of DMSO by 95% (n = 3), and the further addition of ACh did not enhance PGE₂ release. Thus, IDM and ASA decreased the amount PGE₂ released in unstimulated antral mucosa and they eliminated the enhancement of PGE₂ release in ACh-stimulated mucosa. The inhibitory effects of IDM on COX activities appear to be stronger than those of ASA.

The effects of a COX₁-selective inhibitor (SC560, 100 nM) on PGE₂ release from antral mucosa were examined (Fig. 4B). SC560 (100 nM) decreased the amount of PGE₂ released slightly (by 15%) in unstimulated mucosa (0.116 ± 0.006 µg (g tissue)^–1, n = 5). Acetylcholine, however, did not enhance PGE₂ release (0.119 ± 0.010 µg (g tissue)^–1, n = 3). Thus, COX₁-selective inhibitor (SC560, 100 nM) eliminated the enhancement of PGE₂ release in ACh-stimulated mucosa. However, IM (1 µM), unlike ACh, enhanced PGE₂ release to 200% in the presence of SC560 (100 nM; 0.254 ± 0.051 µg (g tissue)^–1, n = 3).

The effects of a COX₂-selective inhibitor (NS398, 20 µM) on PGE₂ release were examined in antral mucosa. NS398 (20 µM) decreased the amount of PGE₂ released by 60% in unstimulated mucosa (0.058 ± 0.010 µg (g tissue)^–1, n = 5), but the further addition of ACh, however, enhanced PGE₂ release to 350% (0.207 ± 0.035 µg (g tissue)^–1, n = 4). Although the amount of ACh-stimulated PGE₂ release with NS398 was smaller than that without NS398 (0.319 ± 0.026 µg (g tissue)^–1), the amounts of ACh-stimulated increase in PGE₂ release were similar in the presence (0.15 µg (g tissue)^–1) or the absence (0.18 µg (g tissue)^–1) of 20 µM NS398. Ionomycin (1 µM) enhanced PGE₂ release in the presence of NS398 (20 µM; 0.179 ± 0.035 µg (g tissue)^–1, n = 3), similar to ACh.

Thus, a COX₁-selective inhibitor decreased basal PGE₂ release only slightly, but eliminated enhancement of PGE₂ release in ACh-stimulated antral mucosa. Ionomycin, however, enhanced PGE₂ release with SC560. In contrast, a COX₂-selective inhibitor (20 µM NS398) decreased basal PGE₂ release, but ACh still enhanced PGE₂ release. Ionomycin also enhanced PGE₂ release in antral mucosa with NS398. Based on these observations, basal PGE₂ release is maintained by COX₂. Cyclo-oxygenase 1, not COX₂, is stimulated by ACh, although both COX₁ and COX₂ are stimulated by ionomycin -induced [Ca²⁺]_i elevation. This suggests that COX₁ and COX₂ may exist in the different cell types. Cyclo-oxygenase 1-containing cells appear to have ACh receptors, but COX₂-containing cells do not.

Effects of arachidonic acid (AA) and phospholipase A₂ (PLA₂) inhibitors

Prostaglandin E₂ is synthesized from arachidonic acid (AA), which in turn is synthesized via phospholipase A₂ (PLA₂). Inhibition of PLA₂ is expected to inhibit PGE₂ synthesis via inhibition of AA accumulation. The effects of N-(p-amylcinnamoyl) anthranilic acid (ACA, 10 µM, an inhibitor of PLA₂) on PGE₂ release were examined. The addition of ACA (10 µM) decreased the amount of PGE₂ released by 70% (n = 3) in unstimulated mucosa, and the further addition of ACh did not enhance PGE₂ release (n = 3; Fig. 5A). Thus, the inhibition of AA accumulation decreased basal PGE₂ release, and the further addition of ACh did not enhance PGE₂ release.

The effects of AA accumulation on PGE₂ release were examined. The addition of AA (2 µM) increased the amount of PGE₂ released to 200% without ACh stimulation (0.272 ± 0.014 µg (g tissue)^–1, n = 5), and ACh enhanced PGE₂ release to 300% (0.421 ± 0.042 µg (g tissue)^–1, n = 6). However, an ACh-stimulated increase in PGE₂ release with AA (0.15 µg (g tissue)^–1) was similar to that without AA (0.153 µg (g tissue)^–1). Indomethacin (10 µM) eliminated the enhancement of PGE₂ release induced by AA and ACh (Fig. 5A).

We also examined the effects of AA on PGE₂ release in SC560-treated antral mucosa (Fig. 5B). The addition of AA increased the amounts of PGE₂ release to 250% with SC560 (0.312 ± 0.078 µg (g tissue)^–1, n = 4); however, the further stimulation with ACh did not enhance PGE₂ release (0.271 ± 0.012 µg (g tissue)^–1 n = 3). Thus, SC560 did not eliminate the enhancement of PGE₂ release induced by AA, but it eliminated the enhancement of PGE₂ release induced by ACh.

We also examined the effects of AA on PGE₂ release in NS398-treated antral mucosa (Fig. 5B). The addition of AA did not enhance PGE₂ release significantly with NS398 (0.080 ± 0.019 µg (g tissue)^–1, n = 3), and the further stimulation with ACh enhanced PGE₂ release to 350% (0.210 ± 0.035 µg (g tissue)^–1, n = 4). But the amounts of PGE₂ released during ACh stimulation with AA addition (0.13 µg (g tissue)^–1) were similar to those without AA addition (0.15 µg (g tissue)^–1) in NS398-treated antral mucosa.

Thus, COX₁ is unlikely to utilize exogenous AA at a low concentration (2 µM), as previously reported (Murakami et al. 1999).

Prostaglandin E₂ release in isolated antral epithelial cells

Prostaglandin E₂ release from isolated antral epithelial cells was examined, since the antral epithelial cells are suggested to express only COX₁ (Iseki, 1995; Sawaoka et al. 1998). In isolated antral epithelial cells, basal PGE₂ release was detected. Figure 6 shows the relative changes in PGE₂ release from antral epithelial cells. Acetylcholine enhanced PGE₂ release by 150% in the isolated antral epithelial cells (n = 5). Indomethacin (10 µM) decreased the amount of PGE₂ released by 85% (n = 3) in unstimulated cells, and the further addition of ACh did not enhance PGE₂ release. SC560 (100 nM) decreased the amount of PGE₂ released by 65% (n = 4), and the further addition of ACh did not enhance PGE₂ release. However, NS398 (20 µM) did not decrease the amount of PGE₂ released (117%, n = 4), and further addition of ACh enhanced PGE₂ release (197%, n = 4). Thus, COX₁, not COX₂, mediates PGE₂ production in isolated antral epithelial cells.

	Discussion

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Recent findings indicate that COX₂ plays crucial roles in the function of the kidney (Harris, 1996), cardiovascular system (McAdam et al. 1999; Peskar et al. 2001; Simmons et al. 2004), brain (Lukiw & Bazan, 1997) and pancreatic islet (Robertson, 1998). In the stomach, however, the physiological roles of COX₂ still remain uncertain, although both COX₁ and COX₂ are required for gastric mucosal defense (Gretzer et al. 1998, 2001; Wallace et al. 2000; Peskar et al. 2001; Simmons et al. 2004). The present study demonstrated that COX₂ produces PGE₂ in unstimulated antral mucosa spontaneously. This is the first report to show that COX₂ maintains the basal PGE₂ release in the antral mucosa. The present study also demonstrated that COX₁-derived PGE₂ is a small proportion of total the PGE₂ pool (approximately 10%) in unstimulated antral mucosa, and that COX₁ activity appears to be low in unstimulated mucosa (at basal [Ca²⁺]_i).

In ACh-stimulated antral mucosa, however, the amount of PGE₂ derived via COX₁ increased to approximately 60% of the total PGE₂ pool, although the amounts of PGE₂ derived from COX₂ were unaffected by ACh. In gastric mucosa, COX₁-derived PGE₂ appears to contribute to the resistance of the antral mucosa to damage induced by HCl and peptic enzymes secreted during ACh stimulation. Our previous report demonstrated that COX₁-derived PGE₂ enhances mucin exocytosis in ACh-stimulated antral mucous cells (Shimamoto et al. 2005).

Cyclo-oxygenase 1 and COX₂ activities were regulated by [Ca²⁺]_i. Acetylcholine, which increases [Ca²⁺]_i via cholinergic receptors (Suzuki et al. 1993; Fujiwara et al. 1999, 2006; Nakahari et al. 2002; Shimamoto et al. 2005), increased the amounts of PGE₂ released via COX₁, not via COX₂, in antral mucosa and antral epithelial cells. This indicates that cholinergic receptors exist in cells containing COX₁. Antral epithelial cells have been shown to stain positively for COX₁ by immunocytochemistry in rats (Iseki, 1995) and guinea-pigs (data not shown). Moreover, ACh-stimulated exocytosis of antral mucous cells was partly inhibited by a COX₁-selective inhibitor (SC560; Shimamoto et al. 2005). Thus, antral epithelial cells synthesize PGE₂ via COX₁ in response to ACh stimulation, although COX₁ activity is low at a low [Ca²⁺]_i.

In contrast, COX₂ generated PGE₂ in unstimulated antral mucosa and was unaffected by ACh. Cyclo-oxygenase 2-containing cells appear to have no cholinergic receptors, suggesting that they are non-epithelial cells, such as interstitial cells, vascular cells or macrophages. In immunohistochemical examinations, the specific immunostaining of COX₂ was undetectable in normal antral mucosa of guinea-pigs (data not shown). Similar observations have already been reported in gastric mucosa of rats and humans (Iseki, 1995; Kargman et al. 1996; Peskar et al. 2001; Sun et al. 2000; Tatsuguchi et al. 2000; Tanigawa et al. 2004). In those reports, however, COX₂ immunoreactivity was detected in interstitial cells, such as macrophages and fibroblasts, in injured gastric mucosa, although it was also detected in gastric epithelial cells. Based on these observations, it is suggested that COX₂ exists at an undetectable concentration in normal gastric mucosa. In antral mucosa, COX₂ may also exist at undetectable concentrations, and this COX₂ in interstitial cells generates PGE₂ in unstimulated antral mucosa. Cyclo-oxygenase 2 activity appears to be high even at a low [Ca²⁺]_i (basal level).

The basal PGE₂ release was 9 ng (g tissue)^–1 min^–1 (30 nM (kg tissue)^–1 min^–1). The half-life of PGE₂ is approximately 30 s in the circulatory system (Fitzpatrick et al. 1980). If the PGE₂ released is replaced by newly formed PGE₂ within 1 min and the extracellular fluid volume is assumed to be 25%, the PGE₂ concentration in the antral mucosa is calculated to be 60 nM (Shimamoto et al. 2005). A PGE₂ concentration of 50–100 nM has been demonstrated to activate exocytotic events (1 event min^–1 per cell) in antral mucous cells, and this appears to supply mucins to the surface mucous gel layer in the resting antral mucosa (Ohnishi et al. 2001). Cyclo-oxygenase 2 was further activated by an increase in [Ca²⁺]_i as shown in Fig. 3B. Under pathophysiological conditions, such as gastritis and gastric ulceration, COX₂-derived PGE₂ may increase, since inflammation may stimulate an increase in [Ca²⁺]_i and induce COX₂ (Sawaoka et al. 1998; Sun et al. 2000; Tatsuguchi et al. 2000; Miyake et al. 2002; Tanigawa et al. 2004). Prostaglandin E₂ released via COX₂ appears to play an important role in mucosal protection or repair under not only physiological but also pathophysiological conditions.

Prostaglandin E₂ release was enhanced by AA, and was inhibited by a PLA₂ inhibitor. This indicates that PLA₂ plays a key role in maintaining PGE₂ release in antral mucosa (Fujiwara et al. 2006). Moreover, PLA₂ is known to be a Ca²⁺-regulated enzyme (Murakami et al. 1999). An increase in [Ca²⁺]_i, which also activates PLA₂, induces AA accumulation in antral epithelial cells. Thus, increases in [Ca²⁺]_i stimulate PGE₂ release via two mechanisms, COX stimulation and AA accumulation.

In contrast, AA exogenously added did not increase the amount of COX₁-derived PGE₂ released and did not enhance it during ACh stimulation. A previous study demonstrated that COX₁ does not utilize exogenous AA at a low concentration, such as 2 µM (Murakami et al. 1999). In antral mucosa, COX₁ is unlikely to utilize exogenous AA at a low concentration (2 µM).

Cyclo-oxygenase 1-derived PGE₂ is believed to maintain the integrity of the gastric mucosal layer under physiological conditions. In contrast, COX₂-derived PGE₂ is believed to contribute to gastric mucosal protection and repair under pathophysiological conditions (Sawaoka et al. 1998; Shigeta et al. 1998; Sun et al. 2000; Tatsuguchi et al. 2000; Peskar et al. 2001; Miyake et al. 2002; Takeeda et al. 2004; Tanigawa et al. 2004). The present study, however, suggests that COX₂-derived PGE₂ maintains the integrity of unstimulated antral mucosa under physiological conditions. There are lines of evidence showing that COX₂ plays crucial roles in the maintenance of the integrity of normal gastric mucosa (Gretzer et al. 1998, 2001; Shigeta et al. 1998; Hawkey, 2000; Miyake et al. 2002; Simmons et al. 2004). In contrast, COX₁-derived PGE₂ also contributes to the resistance of the antral mucosa against damage during ACh stimulation, e.g. when food is present in the stomach (Shimamoto et al. 2005).

Our conclusions are illustrated in Fig. 7. The COX₂-derived PGE₂ released from non-epithelial cells maintains the integrity of unstimulated antral mucosa via a paracrine mechanism. In contrast, the COX₁-derived PGE₂ released from antral epithelial cells maintains the integrity of ACh-stimulated antral mucosa and enhances Ca²⁺-regulated exocytosis via an autocrine mechanism, as previously reported (Nakahari et al. 2002; Shimamoto et al. 2005).

View larger version (82K): [in this window] [in a new window]   Figure 1.  Histological sections of the stripped antral mucosa (A and C) and isolated antral columm cells (B and D) A and B, Haematoxylin and Eosin staining. C and D, Periodic Acid–Shiff (PAS) staining. Scale bars; 100 µm in A and C, 30 µm in B and D.

View larger version (10K): [in this window] [in a new window]   Figure 2.  PGE2 release from guinea-pig antral mucosa A, the amount of PGE2 released from the antral mucosa into the incubation medium increased spontaneously (Control). When DMSO (10 µl) was added as the vehicle control, the amount of PGE2 released also increased; however, each value was not significantly different from each corresonding value in the control presparations. Acetylcholine (10 µM) further enhanced the amount of PGE2 released. *Significantly different from the corresponding values (P < 0.05). B, effects of the dose of ACh. The amounts of PGE2 released during 15 min in response to ACh (0.1–100 µM) were plotted against ACh concentration. *Significantly different from the vehicle control value (P < 0.05, n = 3–5 in each experiment).

View larger version (12K): [in this window] [in a new window]   Figure 3.  Effects of Ca2+ on PGE2 release In control experiments, ACh (10 µM), ionomycin (IM, 1 µM) and thapsigargin (TG, 2 µM) increased the amounts of PGE2 released to a similar extent. Under Ca2+-free conditions, basal PGE2 release was suppressed. Acetylcholine still slightly increased the amount of PGE2 release. The slight increase was completely suppressed by the Ca2+-free solution containing BAPTA AM (25 µM). The amounts of PGE2 released during 15 min are shown. *Significantly different from the vehicle control in each experiment for the value during ACh stimulation in the control experiment (P < 0.05). Significantly different (P < 0.05).

View larger version (11K): [in this window] [in a new window]   Figure 4.  Effects of COX inhibitors A, acetylsalicylic acid (ASA, 10 µM) and indomethacin (IDM, 10 µM) suppressed basal PGE2 release and in the presence of ASA or IDM, ACh does not enhance PGE2 release. B, an inhibitor of COX1 (SC560, 100 nM) decreased basal PGE2 release slightly but significantly (P < 0.05). However, ACh (10 µM) did not further increase PGE2 release, whereas ionomycin (IM, 1 µM) increased it. An inhibitor of COX2 (NS398, 20 µM) decreased basal PGE2 release markedly, whereas ACh (10 µM) or IM (1 µM) increased PGE2 release to a similar extent. The amounts of PGE2 released during 15 min are shown. *Significantly different from the vehicle control in the same experiment (P < 0.05). Significantly different from the vehicle control in the initial, control experiment (P < 0.05).

View larger version (10K): [in this window] [in a new window]   Figure 5.  Effects of arachidonic acid (AA) A, an inhibitor of PLA2 (ACA, 10 µM) decreased basal PGE2 release, and ACh (10 µM) induced no further increase in the amount of PGE2 released. Arachidonic acid (AA, 2 µM) increased basal PGE2 release, and ACh (10 µM) further increased PGE2 release, which was completely suppressed by 10 µM indomethacin (IDM). B, in the presense of SC560 (100 nM), AA (2 µM) increased basal PGE2 release markedly, but ACh induced no further increase in the amount of PGE2 released. In the presence of NS398 (20 µM), AA (2 µM) did not increase basal PGE2 release, but ACh (10 µM) increased the amount of PGE2 released. The amounts of PGE2 released during 15 min are shown. *Significantly different from the vehicle control in the same experiment (P < 0.05). Significantly different from the vehicle control in the initial, control experiment (P < 0.05). Paired values marked by • are significantly different (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Figure 6.  PGE2 release from isolated epithelial cells In isolated epithelial cells, IDM or SC560 inhibited basal PGE2 release and ACh-stimulated PGE2 release. However, NS398 had no effects on the amounts of PGE2 released. *Significantly different from the vehicle control in the same experiment (P < 0.05). Significantly different from the vehicle control in the initial, control experiment (P < 0.05).

View larger version (35K): [in this window] [in a new window]   Figure 7.  PGE2 release in antral mucosa

	Footnotes

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	Acknowledgements

 
We thank Drs S. Fujiwara and M. Kato (Department of Physiology, Osaka Medical College) for their technical assistance in cell preparations. This work was supported by a research project (Nakahari Project) of the Central Research Laboratory of Osaka Medical College.

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